Autism spectrum disorder (ASD) is a common and debilitating neurodevelopmental disorder. Although there is strong evidence for a genetic contribution to ASD, the isolation of specific causative genetic defects has been difficult. Our previous research has focused on the homeobox transcription factor ENGRAILED 2 (EN2). We have demonstrated consistent association for two intronic EN2 SNPs, rs1861972 and rs1861973, in three separate datasets. These findings determined that EN2 is a likely ASD susceptibility gene. LD mapping and re-sequencing identified the associated A-C rs1861972-rs1816973 haplotype as a candidate disease allele. Functional studies have now demonstrated that the EN2 intron acts a transcriptional repressor and that the A-C haplotype results in a weaker repressor compared to the non-associated G-T haplotype. EMSAs have determined that DNA binding proteins specifically interact with the associated alleles for both SNPs, providing a mechanism for the observed functional difference. These studies have identified the A-C haplotype as the first common risk factor for autism. The goal of this proposal is to generate a mouse model for the EN2 risk allele by using recombinase- mediated genomic replacement (RMGR). RMGR is preferred over standard targeting approaches because large segments of the mouse genome (>100kb) can be replaced with the syntenic human region. This reduces concerns about the proper regulation of the human gene. Our plan is to use RMGR to replace ~72kb of the mouse En2 locus with the syntenic human region and to generate knock-in lines for both the associated A-C and the non-associated G-T haplotypes. An IRES:red fluorescent protein (DsRed-E5/pTIMER) will also be used to modify the EN2 locus so that subtle effects of the risk allele on levels and spatial/temporal expression can be identified. The knock-ins will then be examined to determine the developmental cell types and ages in which the EN2 risk allele is functional. Studies with the En2 knock-out have uncovered anatomical, developmental and neurochemical phenotypes relevant to ASD. The same analysis will be repeated for the knock-ins as a first step in determining the cellular pathways affected by the risk allele. This information will be critical in the development of better diagnoses, treatments and preventions for ASD.